Despite significant advances in the treatment, development of resistance to conventional chemotherapy remains an unresolved problem. Consequently an in-depth understanding of the mechanisms underlying the development of chemoresistance is of utmost importance for improving the therapeutic regimens. Recent evidence indicates that epithelial to mesenchymal transition (EMT), the transdifferentiation program involved in tumor invasion and metastasis, may also contribute to chemoresistance. However, the importance of EMT in vivo is fiercely debated due to the inability of tracking EMT events during tumor progression. To solve this critical issue, we have established a novel EMT lineage tracing system in breast cancer by combining multiple transgenic models (MMTV-PyMT/Fsp1-Cre/Rosa26-GRFP). In this model, mesenchymal- specific Cre-mediated recombination initiates a permanent switch of fluorescent markers in tumor cells undergoing EMT, which enable us to track the EMT tumor cells throughout the breast tumor progression. The initial studies with this model found that within a predominantly epithelial primary tumor, a small portion (approx. 2%) of tumor cells underwent EMT. Strikingly, these EMT cells did not give rise to metastasis as the metastases were mainly comprised of non-EMT tumor cells (no fluorescence switch). Importantly, we discovered that EMT tumor cells significantly contributed to lung metastasis after chemotherapy. The EMT tumor cells survived chemotherapy due to reduced proliferation, apoptotic tolerance, and elevated expression of chemoresistance- related genes. Inhibiting EMT by overexpressing miR-200 family abrogated this resistance. These results provide direct evidence of EMT in chemoresistance and open the window to investigate EMT-targeting strategies as a means to overcome chemoresistance in breast cancer. In this proposal, we will take advantage of the EMT lineage tracing model, to further investigate a series of rationally guided anti-EMT approaches such as targeting EMT-induced autocrine/paracrine signals (Aim 1) and EMT-promoting transcription factors (Aim 2). We will use both genetic and pharmacological approaches to evaluate their importance in EMT-mediated chemoresistance. Moreover, based on our preliminary transcriptome analysis we will generate a CRISPR gRNA library targeting the differentially expressed genes in EMT tumor cells. The EMT lineage tracing model will provides a unique platform for the CRISPR screen to identify novel therapeutic targets for the EMT-mediated chemoresistance (Aim 3). At the end, we will validate our findings in human breast cancer and evaluate efficacy of combination therapies in a series of metastatic breast cancer patient-derived xenograft (PDX) models (Aim 4). We envision that this mechanism-based targeting of EMT- mediated chemoresistance will constitute feasible strategies for breast cancer therapy.